Capillary isoelectric focusing (cIEF), which was first demonstrated by Hjertén and Zhu (J. Chomatogr. 1985, 346, 265), has proven to be a powerful method for protein and peptide analysis. cIEF is typically performed as a two-step process, with a focusing (separation) step followed by a mobilization step. During focusing, proteins or peptides are separated based on their isoelectric point (pI). Optical detection can be used to image the focused bands within the separation capillary.
Alternatively, the focused sample is mobilized to a detector placed at the capillary's distal end. Two kinds of mobilization approaches are typically employed. Hydraulic mobilization is produced using pressure, vacuum, or syphon. Alternatively, chemical mobilization, as first proposed by Hjertén (J. Chromatogr. 1987, 387, 127-138), is performed by changing the catholyte to lower pH buffer. Chemical mobilization avoids Taylor dispersion due to laminar flow during pressure mobilization.
cIEF has been coupled to a range of detectors, including UV absorbance, laser-induced fluorescence, and mass spectrometry. Mass spectrometry provides information-rich identification such as accurate molecular weight and fragmentation information. Although there are some challenges for on-line coupling cIEF to mass spectrometry, several reports have analyzed complex mixtures of proteins or peptides.
However, earlier cIEF-MS experimental methods employed chemical mobilization that required significant manual manipulation of the capillary. Focusing was performed off-line using appropriate buffers in dedicated reservoirs. After focusing, the capillary was manually inserted into the emitter, where the acidic sheath electrolyte served as mobilization buffer and generated chemical mobilization to drive the samples to the mass spectrometer. This manual operation is time-consuming, tedious, frustrates automation, and limits the practical application of this technique.
Accordingly, there is a need to perform cIEF-MS experiments that do not require cumbersome and significant manual manipulation of the capillary for off-line focusing and chemical mobilization.